This application is in response to PAR-14-279 Discovery of in vivo Chemical Probes (R01) and targets arthritis, musculoskeletal and skin diseases. We propose to validate S1PR3 as a mechanistic target for arthritis and fibrosis. The class A GPCR superfamily is highly productive for drug discovery. The sphingosine- 1-phosphate receptor family consists of 5 GPCRs, S1PR1, S1PR2, S1PR3, S1PR4 and S1P5 and regulates multiple biological responses including lymphocyte trafficking, vascular tone, cell differentiation and survival. The literature and our preliminary data support a role for S1P in inflammation and fibrosis. Agonists of S1PR3 have fibrosis as the dose-limiting toxicity across multiple species including primates. Also, S1PR3 enhances cytokine production in response to LPS, and both deletion of S1PR3 or chemical antagonism blunts cytokine amplification and inhibits fibrosis. The proliferation, migration, secretion of extra-cellular matrix and inflammator cytokines by Rheumatoid arthritis fibroblast-like-synoviocytes (RA-FLS) are thought to be central in disease progression and are dependent on both TNF and S1P. S1PR3 expression is elevated in RA-FLS. Genetic deletion of Sphingosine kinase 1 in the TNF-?-induced arthritis mouse reduced symptoms including synovial inflammation and joint erosion. S1P is also elevated in the synovial fluid of RA patients. We propose to validate S1PR3 as a therapeutic target by identifying potent, selective, bioavailable antagonists of the S1PR3 receptor and demonstrating proof-of-concept in vitro and in vivo. These goals will be approached through two Specific Aims: Aim 1: Develop and optimize S1PR3-selective ligands with improved potency, selectivity and pharmacokinetic properties. Current S1P receptor ligands will be optimized for potency and functional selectivity for S1PR3 versus other S1P receptor family members; S1P1, S1P2, S1P4 and S1P5. Physicochemical properties of S1PR3 selective antagonist compounds including microsome stability, solubility, total polar surface area and pharmacokinetics properties (joint distribution) will be optimized for in vivo use. Aim 2: Pharmacological and functional characterization of S1PR3 antagonists in vitro and in vivo. This project will deliver new selective potent and bioavailable probes that specifically modulate S1PR3 signaling. Such compounds will facilitate validation of S1PR3 as a drug target for rheumatoid arthritis. We will test compounds in vitro for their ability to inhibit cell proliferation, migration and cytokine secretion and in vivo to reduce RA symptoms and outcomes in TNFdeltaARE model.